1. Technical Field
The present disclosure relates to a method for analyzing nucleic acid targets.
2. Description of the Related Art
As a basic technique for realizing this, a method of amplifying a desired nucleic acid target in nucleic acid such as DNA or RNA to such a quantity that is required for detection with a technique called PCR (polymerase chain reaction), and detecting the amplified target is generally known. As an advanced form of this technique, a quantitative analytic technique called qPCR (quantitative PCR) is generally used. Such a quantitative analytic technique for a nucleic acid target is introduced into a small reactor in a microfluidic chip.
PTL 1 discloses a basic method for realizing qPCR for quantitative analysis of gene. PTL 1 discloses a method that includes bringing a sample of single-stranded DNA into contact with an oligo nucleotide having a sequence that is complementary to a first region of the sequence chain of a target DNA (shorter DNA/RNA sequence), and with a labeled oligonucleotide including a sequence that is complementary to a second region of the sequence chain of the same target DNA, to form a mixture of double-stranded complex under the condition that causes hybridization; cutting the annealed labeled oligonucleotide by 5′→3′ nuclease activity to liberate the labeled fragment; and detecting the liberated labeled fragment. When a fluorescent dye and a quencher are used as the labeled fragment of the labeled oligonucleotide, fluorescence is emitted only after the labeled fragment is liberated. Therefore, the intensity of fluorescence increases by repetition of this process. In general, the condition of causing hybridization, the condition of causing nuclease activity and so on are realized by exposing an analyte under a certain temperature condition. Accordingly, repeating the aforementioned process means repeating predetermined temperature rise and fall (temperature cycle) on the analyte. By detecting the intensity of fluorescence with a photo detector, and examining the relation between the number of repetition of the aforementioned process (the number of temperature cycles) and the intensity of fluorescence, it is possible to analyze the degree of the objective sequence chain of the target DNA contained in the analyte. In the case of detecting targets of multiple sites per analyte, it becomes possible to conduct separation and analysis according to the difference in fluorescent wavelength in the photo detector by preparing labeled oligonucleotides that are complementary to the respective sequence chains, and differentiating the material and wavelength of the fluorescent dye which is to be a label among the different labeled oligonucleotides.
PTL 2 discloses one exemplary method for realizing the technique of quantitatively analyzing gene. In particular, an improved technique of a high throughput assay based on the emulsification technique is disclosed. Disclosed is a method of producing drops each functioning as an independent reaction chamber for biochemical reaction by the emulsification technique, and treating and assaying individual subcomponents (cell, nucleic acid, protein etc.) by using these drops.
By suspending water drops composed of DNA/RNA and so on in oil, it is possible to produce an oil-in-water emulsion. By stabilizing the emulsion with a surfactant, it is possible to reduce or eliminate binding of drops during heating, cooling and transportation, and thus it is possible to carry out a temperature cycling conducted in the PCR technique or the like. Therefore, amplification of single copy of a nucleic acid target molecule in drops by PCR is conducted by using an emulsion. Among these drops, drops that are positive to a specific target are analyzed according to the Poisson statistics, and the concentration of the target in the sample can be estimated. In the assay using drops, whether the reaction such as amplification occurs can be recognized by using one or more than one kinds of fluorescent substances as a label in drops. By generating liquid drops, reacting the drops, and measuring light emitted from each drop, it may be possible to determine whether the target exists in the drop. By assigning discriminable different kinds of fluorescent substances to different targets respectively, it is possible to determine whether multiple different targets exist in each drop. In discriminating the multiple different targets as described above, it is general to employ a technique of using multiple kinds of fluorescent substances, namely dye materials emitting fluorescence of different wavelength, and discriminating the targets according to the fluorescence wavelength. PTL 2 discloses a method of using two kinds of fluorescent dyes and detecting them discriminately. Detection systems (each including a light source and a detector) respectively corresponding to a first dye and a second dye are provided, and drops are detected alternately by the detection system corresponding to the first dye and the detection system corresponding to the second dye while the drops pass through the detection area of the flow channel.